Ferriphaselus amnicola strain GF-20, a new iron- and thiosulfate-oxidizing bacterium isolated from a hard rock aquifer.

Ferriphaselus amnicola GF-20 is the first Fe-oxidizing bacterium isolated from the continental subsurface. It was isolated from groundwater circulating at 20 m depth in the fractured-rock catchment observatory of Guidel-Ploemeur (France). Strain GF-20 is a neutrophilic, iron- and thiosulfate-oxidizer and grows autotrophically. The strain shows a preference for low oxygen concentrations, which suggests an adaptation to the limiting oxygen conditions of the subsurface. It produces extracellular stalks and dreads when grown with Fe(II) but does not secrete any structure when grown with thiosulfate. Phylogenetic analyses and genome comparisons revealed that strain GF-20 is affiliated with the species F. amnicola and is strikingly similar to F. amnicola strain OYT1, which was isolated from a groundwater seep in Japan. Based on the phenotypic and phylogenetic characteristics, we propose that GF-20 represents a new strain within the species F. amnicola.

Effect of arsenite and growth in biofilm conditions on the evolution of Thiomonas sp. CB2.

Thiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjugative element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments.

Genome reconstruction reveals distinct assemblages of Gallionellaceae in surface and subsurface redox transition zones.

Fe-oxidizing bacteria of the family Gallionellaceae are major players in the Fe biogeochemical cycle in freshwater. These bacteria thrive in redox transition zones where they benefit from both high Fe concentrations and microaerobic conditions. We analysed the Gallionellaceae genomic diversity in an artesian hard-rock aquifer where redox transition zones develop (i) in the subsurface, where ancient, reduced groundwater mixes with recent oxygenated groundwater, and (ii) at the surface, where groundwater reaches the open air. A total of 15 new draft genomes of Gallionellaceae representing to 11 candidate genera were recovered from the two redox transition zones. Sulfur oxidation genes were encoded in most genomes while denitrification genes were much less represented. One genus dominated microbial communities belowground and we propose to name it 'Candidatus Houarnoksidobacter'. The two transition zones were populated by completely different assemblages of Gallionellaceae despite the almost constant upward circulation of groundwater between the two zones. The processes leading to redox transition zones, oxygen diffusion at the surface or groundwater mixing in subsurface, appear to be a major driver of the Gallionellaceae diversity.

Kinetic Study on Clogging of a Geothermal Pumping Well Triggered by Mixing-Induced Biogeochemical Reactions.

The sustainability of ground-source geothermal systems can be severely impacted by microbially mediated clogging processes. Biofouling of water wells by hydrous ferric oxide is a widespread problem. Although the mechanisms and critical environmental factors associated with clogging development are widely recognized, effects of mixing processes within the wells and time scales for clogging processes are not well characterized. Here we report insights from a joint hydrological, geochemical, and metagenomics characterization of a geothermal doublet in which hydrous ferric oxide and hydrous manganese oxide deposits had formed as a consequence of mixing shallow groundwater containing dissolved oxygen and nitrate with deeper, anoxic groundwater containing dissolved iron (FeII) and manganese (MnII). Metagenomics identify distinct bacteria consortia in the pumping well oxic and anoxic zones, including autotrophic iron-oxidizing bacteria. Batch mixing experiments and geochemical kinetics modeling of the associated reactions indicate that FeII and MnII oxidation are slow compared to the residence time of water in the pumping well; however, adsorption of FeII and MnII by accumulated hydrous ferric oxide and hydrous manganese oxide in the well bore and pump riser provides "infinite" time for surface-catalyzed oxidation and a convenient source of energy for iron-oxidizing bacteria, which colonize the surfaces and also catalyze oxidation. Thus, rapid clogging is caused by mixing-induced redox reactions and is exacerbated by microbial activity on accumulated hydrous oxide surfaces.

Correction to: Adaptation in toxic environments: comparative genomics of loci carrying antibiotic resistance genes derived from acid mine drainage waters.

The original version of this article unfortunately contains a mistake.

Adaptation in toxic environments: comparative genomics of loci carrying antibiotic resistance genes derived from acid mine drainage waters.

Several studies have suggested the existence of a close relationship between antibiotic-resistant phenotypes and resistance to other toxic compounds such as heavy metals, which involve co-resistance or cross-resistance mechanisms. A metagenomic library was previously constructed in Escherichia coli with DNA extracted from the bacterial community inhabiting an acid mine drainage (AMD) site highly contaminated with heavy metals. Here, we conducted a search for genes involved in antibiotic resistance using this previously constructed library. In particular, resistance to antibiotics was observed among five clones carrying four different loci originating from CARN5 and CARN2, two genomes reconstructed from the metagenomic data. Among the three CARN2 loci, two carry genes homologous to those previously proposed to be involved in antibiotic resistance. The third CARN2 locus carries a gene encoding a membrane transporter with an unknown function and was found to confer bacterial resistance to rifampicin, gentamycin, and kanamycin. The genome of Thiomonas delicata DSM 16361 and Thiomonas sp. X19 were sequenced in this study. Homologs of genes carried on these three CARN2 loci were found in these genomes, two of these loci were found in genomic islands. Together, these findings confirm that AMD environments contaminated with several toxic metals also constitute habitats for bacteria that function as reservoirs for antibiotic resistance genes.

Comparison of biofilm formation and motility processes in arsenic-resistant Thiomonas spp. strains revealed divergent response to arsenite.

Bacteria of the genus Thiomonas are found ubiquitously in arsenic contaminated waters such as acid mine drainage (AMD), where they contribute to the precipitation and the natural bioremediation of arsenic. In these environments, these bacteria have developed a large range of resistance strategies among which the capacity to form particular biofilm structures. The biofilm formation is one of the most ubiquitous adaptive response observed in prokaryotes to various stresses, such as those induced in the presence of toxic compounds. This study focused on the process of biofilm formation in three Thiomonas strains (CB1, CB2 and CB3) isolated from the same AMD. The results obtained here show that these bacteria are all capable of forming biofilms, but the architecture and the kinetics of formation of these biofilms differ depending on whether arsenite is present in the environment and from one strain to another. Indeed, two strains favoured biofilm formation, whereas one favoured motility in the presence of arsenite. To identify the underlying mechanisms, the patterns of expression of some genes possibly involved in the process of biofilm formation were investigated in Thiomonas sp. CB2 in the presence and absence of arsenite, using a transcriptomic approach (RNA-seq). The findings obtained here shed interesting light on how the formation of biofilms, and the motility processes contribute to the adaptation of Thiomonas strains to extreme environments.

Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage.

The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ.

Thiomonas sp. CB2 is able to degrade urea and promote toxic metal precipitation in acid mine drainage waters supplemented with urea.

The acid mine drainage (AMD) in Carnoulès (France) is characterized by the presence of toxic metals such as arsenic. Several bacterial strains belonging to the Thiomonas genus, which were isolated from this AMD, are able to withstand these conditions. Their genomes carry several genomic islands (GEIs), which are known to be potentially advantageous in some particular ecological niches. This study focused on the role of the "urea island" present in the Thiomonas CB2 strain, which carry the genes involved in urea degradation processes. First, genomic comparisons showed that the genome of Thiomonas sp. CB2, which is able to degrade urea, contains a urea genomic island which is incomplete in the genome of other strains showing no urease activity. The urease activity of Thiomonas sp. CB2 enabled this bacterium to maintain a neutral pH in cell cultures in vitro and prevented the occurrence of cell death during the growth of the bacterium in a chemically defined medium. In AMD water supplemented with urea, the degradation of urea promotes iron, aluminum and arsenic precipitation. Our data show that ureC was expressed in situ, which suggests that the ability to degrade urea may be expressed in some Thiomonas strains in AMD, and that this urease activity may contribute to their survival in contaminated environments.

Adaptation in Toxic Environments: Arsenic Genomic Islands in the Bacterial Genus Thiomonas.

Acid mine drainage (AMD) is a highly toxic environment for most living organisms due to the presence of many lethal elements including arsenic (As). Thiomonas (Tm.) bacteria are found ubiquitously in AMD and can withstand these extreme conditions, in part because they are able to oxidize arsenite. In order to further improve our knowledge concerning the adaptive capacities of these bacteria, we sequenced and assembled the genome of six isolates derived from the Carnoulès AMD, and compared them to the genomes of Tm. arsenitoxydans 3As (isolated from the same site) and Tm. intermedia K12 (isolated from a sewage pipe). A detailed analysis of the Tm. sp. CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes. We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7). Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.

Toxic metal resistance in biofilms: diversity of microbial responses and their evolution.

Since biofilms are an important issue in the fields of medicine and health, several recent microbiological studies have focused on their formation and their contribution to toxic compound resistance mechanisms. In this review, we describe how metals impact biofilm formation and resistance, and how biofilms can help cells resist toxic metals. First, the organic matrix acts as a barrier isolating the cells from many environmental stresses. Secondly, the metabolism of the cells changes, and a slowly-growing or non-growing sub-population of cells known as persisters emerges. Thirdly, in the case of multispecies biofilms, metabolic interactions are developed, allowing cells to be more persistent or to have greater capacity to survive than a single species biofilm. Finally, we discuss how the high density of the cells may promote horizontal gene transfer processes, resulting in the acquisition of new features. All these crucial mechanisms enable microorganisms to survive and colonize toxic environments, and probably accelerate ongoing evolutionary processes.

'Ménage à trois': a selfish genetic element uses a virus to propagate within Thermotogales.

Prokaryotic viruses play a major role in the microbial ecology and evolution. However, the virosphere associated with deep-sea hydrothermal ecosystems remains largely unexplored. Numerous instances of lateral gene transfer have contributed to the complex and incongruent evolutionary history of Thermotogales, an order well represented in deep-sea hydrothermal vents. The presence of clustered regularly interspaced short palindromic repeats (CRISPR) loci has been reported in all Thermotogales genomes, suggesting that these bacteria have been exposed to viral infections that could have mediated gene exchange. In this study, we isolated and characterized the first virus infecting bacteria from the order Thermotogales, Marinitoga piezophila virus 1 (MPV1). The host, Marinitoga piezophila is a thermophilic, anaerobic and piezophilic bacterium isolated from a deep-sea hydrothermal chimney. MPV1 is a temperate Siphoviridae-like virus with a 43.7 kb genome. Surprisingly, we found that MPV1 virions carry not only the viral DNA but preferentially package a plasmid of 13.3 kb (pMP1) also carried by M. piezophila. This 'ménage à trois' highlights potential relevance of selfish genetic elements in facilitating lateral gene transfer in the deep-sea biosphere.